The vaginal epithelium plays an important role in the initial response to microorganisms found in the vaginal tract. The ability of a vaginally-isolated strain of E. faecalis
to inhibit TSST-1-induced IL-8 from the vaginal epithelium may prove to be significant since we have previously shown that “outside-in” signaling resulting in superantigen-induced inflammation may play a role in the development of TSS from the vaginal mucosa 
. We have shown that TSST-1-induced inflammation initiated by epithelial cell production of cytokines and chemokines may open the mucosal barrier and recruit adaptive immune cells to the submucosa so that the superantigen can interact with these cells in order to initiate the cascade of events that leads to systemic TSS 
. This is a similar paradigm to that proposed for HIV: the virus signals through vaginal epithelial cell production of cytokines/chemokines to recruit its target cells to the submucosa in order to initiate an active infection 
. This “outside-in” signaling may prove to be even more important as we continue to characterize how other mucosal pathogens initiate systemic infections. Indeed, three vaginal pathogens initially examined here, C. albicans, G. vaginalis
, and N. gonorrhoeae
, all induced IL-8 responses from HVECs, but those responses were blocked in the presence of E. faecalis
MN1. Therefore, these anti-inflammatory tetramic acid compounds, which can be found naturally produced by microbes in the human vaginal tract, may be useful as therapeutics to block the development of TSS and other systemic infections that start at the vaginal surface.
Lactic acid is a secreted factor that is known to interfere with colonization by potential vaginal pathogens. Although the pH of the tissue culture medium was significantly lower when E. faecalis MN1 was incubated with the HVECs after 3 h, neutralization of the pH at 3 h did not interfere with IL-8 inhibition. This neutralization lasted until the end of the experiment (3 h later), indicating that lactic acid does not play a role in IL-8 inhibition. It is possible, however, that a decrease in pH due to lactic acid changes the ability of the HVECs to respond to TSST-1 and that this happens rapidly after the addition of enterococci to the medium, so that neutralization after 3 h does not matter.
Our studies with E. faecalis
MN1 show for the first time that, in addition to lactic acid, the organism produces two tetramic acid molecules, at least one highly similar to reutericyclin, which was originally isolated from Lactobacillus reuteri
from a sourdough bread culture 
. Reutericyclin has been shown to act as a proton-ionophore to disrupt the pH gradient of bacterial membranes, causing growth inhibition of Gram-positive organisms (other lactobacilli, staphylococci, and enterococci). Gram-negative organisms appear to be protected due to their lipopolysaccharide outer membranes; however mutation of the outer membrane confers susceptibility to reutericyclin 
. Reutericyclin is also anti-inflammatory 
. Recently, we postulated that another anti-inflammatory compound, GML, acted to inhibit Gram-positive bacterial growth in a mechanism similar to that of tetramic acids 
. Indeed, GML has structural similarities to tetramic acids, and the fatty acid monoester appears to function as a quorum sensing molecule for Pseudomonas aeruginosa
, another organism that produces tetramic acids and is resistant to GML 
. Interestingly, we have shown that GML also inhibits cytokine and chemokine responses from human vaginal epithelial cells in response to TSST-1 and HIV-1 
. Thus, we propose that the two tetramic acid, reutericyclin-like compounds made by E. faecalis
MN1 exhibit anti-inflammatory properties on vaginal epithelial cells through their potential ability to alter signal transduction across eukaryotic membranes in addition to bacterial membranes.
MN1 filter-sterilized culture supernates and low molecular weight fractions significantly inhibited T cell proliferation caused by TSST-1, likely due to the presence of tetramic acids. This effect is also observed with GML 
. It is interesting to note that the highest concentrations of culture supernates also prevented proliferation of PBMCs in the absence of TSST-1, and the highest concentrations of low molecular weight fractions reduced PBMC proliferation below the cells only control. This confirms that the tetramic acid, reutericyclin-like compounds have a more generalized immunomodulatory effect, and their actions are not restricted to epithelial cells. These findings are also significant because they suggest that T cells recruited into the submucosa may be inhibited from proliferation due to TSST-1 or other factors such as HIV. It is well known that activated T cells are more easily infected by HIV than unactivated cells. Thus, tetramic acids (and GML) may help prevent infections in three ways: 1) antimicrobial activity; 2) inhibition of epithelial cell production of cytokines and chemokines; and 3) inhibition of activation of T cells.
The concept of reducing innate immune responses to vaginal pathogens in order to prevent the progression of disease may be counterintuitive, but in many cases too much immune system activation can be detrimental to the host. An exuberant immune response can lead to tissue destruction, which contributes to a pathogen's ability to invade deeper host tissues; alternatively, what should be a protective immune response can lead to the recruitment of host cells that can subsequently be infected by some pathogens. A balance may be necessary in the vaginal microenvironment that allows the host to respond appropriately to harmful pathogens without contributing to the progression of disease, and anti-inflammatory compounds such as these naturally secreted reutericyclin-like, tetramic acid compounds may confer unique benefits in establishing a healthy microbial equilibrium.